Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor

ABSTRACT

A method for the direct treatment towards the specific sites of a disease is disclosed. This method is based on the delivery of proteins by catheterization to discrete blood vessel segments using genetically modified or normal cells or other vector systems. Endothelial cells expressing recombinant therapeutic agent or diagnostic proteins are situated on the walls of the blood vessel or in the tissue perfused by the vessel in a patient. This technique, provides for the transfer of cells or vectors and expression of recombinant genes in vivo and allows the introduction of proteins of therapeutic or diagnostic value for the treatment of diseases.

This is a continuation-in-part of U.S. patent application Ser. No.07/724,509, filed on Jun. 28, 1991, now pending, which is acontinuation-in-part of U.S. patent application Ser. No. 07/331,336,filed on Mar. 31, 1989, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the treatment of diseases by thesite-specific instillation or transformation of cells and kits therefor.The present invention also relates to a method for modulating the immunesystem of an animal by the in vivo introduction of recombinant genes.

2. Discussion of the Background

The effective treatment of many acquired and inherited diseases remainsa major challenge to modern medicine. The ability to deliver therapeuticagents to specific sites in vivo would an asset in the treatment of,e.g., localized diseases. In addition the ability to cause a therapeuticagent to perfuse through the circulatory system would be effective forthe treatment of, e.g., inherited diseases and acquired diseases orcancers.

For example, it would be desirable to administer in a steady fashion anantitumor agent or toxin in close proximity to a tumor. Similarly, itwould be desirable to cause a perfusion of, e.g., insulin in the bloodof a person suffering from diabetes. However, for many therapeuticagents there is no satisfactory method of either site-specific orsystemic administration.

In addition, for many diseases, it would be desirable to cause, eitherlocally or systemically, the expression of a defective endogenous gene,the expression of a exogenous gene, or the suppression of an endogenousgene. Again, these remain unrealized goals.

In particular, the pathogenesis of atherosclerosis is characterized bythree fundamental biological processes. These are: 1) proliferation ofintimal smooth muscle cells together with accumulated macrophages; 2)formation by the proliferated smooth muscle cells of large amounts ofconnective tissue matrix; and 3) accumulation of lipid, principally inthe form of cholesterol esters and free cholesterol, within cells aswell as in surrounding connective tissue.

Endothelial cell injury is an initiating event and is manifested byinterference with the permeability barrier of the endothelium,alterations in the nonthrombogenic properties of the endothelialsurface, and promotion of procoagulant properties of the endothelium.Monocytes migrate between endothelial cells, become active as scavengercells, and differentiate into macrophages.

Macrophages then synthesize and secrete growth factors includingplatelet derived growth factor (PDGF), fibroblast growth factor (FGF),epidermal growth factor (EGF), and transforming growth factor alpha(TGF-α). These growth factors are extremely potent in stimulating themigration and proliferation of fibroblasts and smooth muscle cells inthe atherosclerotic plaque. In addition, platelets may interact with theinjured endothelial cell and the activated macrophage to potentiate theelaboration of growth factors and thrombus formation.

Two major problems in the clinical management of coronary artery diseaseinclude thrombus formation in acute myocardial ischemia and restenosisfollowing coronary angioplasty (PTCA). Both involve common cellularevents, including endothelial injury and release of potent growthfactors by activated macrophages and platelets. Coronary angioplastyproduces fracturing of the atherosclerotic plaque and removal of theendothelium. This vascular trauma promotes platelet aggregation andthrombus formation at the PTCA site. Further release of mitogens fromplatelets and macrophages, smooth muscle cell proliferation and monocyteinfiltration result in restenosis.

Empiric therapy with antiplatelet drugs has not prevented this problem,which occurs in one-third of patients undergoing PTCA. A solution torestenosis is to prevent platelet aggregation, thrombus formation, andsmooth muscle cell proliferation.

Thrombus formation is also a critical cellular event in the transitionfrom stable to unstable coronary syndromes. The pathogenesis most likelyinvolves acute endothelial cell injury and/or plaque rupture, promotingdysjunction of endothelial cell attachment, and leading to the exposureof underlying macrophage foam cells. This permits the opportunity forcirculating platelets to adhere, aggregate, and form thrombi.

The intravenous administration of thrombolytic agents, such as tissueplasminogen activator (tPA) results in lysis of thrombus inapproximately 70% of patients experiencing an acute myocardialinfarction. Nonetheless, approximately 30% of patients fail toreperfuse, and of those patients who undergo initial reperfusion of theinfarct related artery, approximately 25% experience recurrentthrombosis within 24 hours. Therefore, an effective therapy forrethrombosis remains a major therapeutic challenge facing the medicalcommunity today.

As noted above, an effective therapy for rethrombosis is by far not theonly major therapeutic challenge existing today. Others include thetreatment of other ischemic conditions, including unstable angina,myocardial infarction or chronic tissue ischemia, or even the treatmentof acquired and inherited diseases or cancers. These might be treated bythe effective administration of anticoagulants, vasodilatory,angiogenic, growth factors or growth inhibitors to a patient. Thus,there remains a strongly felt need for an effective therapy in all ofthese clinical settings.

In addition, it is desirable to be able to modulate the immune system ofan animal. In particular, much effort has been directed toward thedevelopment of vaccines to provide immunological protection frominfection. However, the development of safe vaccines which can bereadily administered to large numbers of patients is problematic, andfor many diseases, such as, e.g., AIDS, no safe and effective vaccine isas yet available. Further, it is also sometimes desirable tospecifically suppress an animals immune response to prevent rejection ofa transplant. Efforts to suppress transplant rejection have resulted inthe development of drugs which result in a general suppression of theimmune response, rather than specific supression to transplantationantigens, and such drugs are not always effective. Thus, there remains aneed for a method to modulate the immune system of an animal.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a novelmethod for the site-specific administration of a therapeutic agent.

It is another object of the present invention to provide a method forthe perfusion of a therapeutic agent in the blood stream of a patient.

It is another object of the present invention to provide a method forcausing the expression of an exogenous gene in a patient.

It is another object of the present invention to provide a method forcausing the expression of a defective endogenous gene in a patient.

It is another object of the present invention to provide a method forsuppressing the expression of an endogenous gene in a patient.

It is another object of the present invention to provide a method forsite-specifically replacing damaged cells in a patient.

It is another object of the present invention to provide a method forthe treatment of a disease by causing either the site-specificadministration of a therapeutic agent or the perfusion of a therapeuticagent in the bloodstream of a patient.

It is another object of the present invention to provide a method forthe treatment of a disease by causing either the expression of anexogenous gene, the expression of a defective endogenous gene, or thesuppression of the expression of an endogenous gene in a patient.

It is another object of the present invention to provide a method forthe treatment of a disease by site-specifically replacing damaged cellsin a patient.

It is another object of the present invention to provide a kit forsite-specifically instilling normal or transformed cells in a patient.

It is another object of the present invention to provide a kit forsite-specifically transforming cells in vivo.

It is another object of the present invention to provide a method formodulating the immune system of an animal.

It is another object of the present invention to provide a method formodulating the immune system of an animal to sensitize the animal to aforeign molecule.

It is another object of the present invention to provide a method tostimulate the immune system of an animal to reject proteins in order toprotect against infection by a microorganism or virus.

It is another object of the present invention to provide a method formodulating the immune system of an animal to tolerize the animal to aforeign molecule.

It is another object of the present invention to provide a method formodulating the immune system of an animal to reduce the tendency toreject a transplant.

It is another object of the present invention to provide a novel kit fortransforming cells by systemic administration in vivo.

These and other objects of this invention which will become apparentduring the course of the following detailed description of the inventionhave been discovered by the inventors to be achieved by (a) a methodwhich comprises either (i) site-specific instillation of either normal(untransformed) or transformed cells in a patient or (ii) site-specifictransformation of cells in a patient and (b) a kit which contains acatheter for (i) site-specific instillation of either normal ortransformed cells or (ii) site-specific transformation of cells.

Site-specific instillation of normal cells can be used to replacedamaged cells, while instillation of transformed cells can be used tocause the expression of either a defective endogenous gene or anexogenous gene or the suppression of an endogenous gene product.Instillation of cells in the walls of the patient's blood vessels can beused to cause the steady perfusion of a therapeutic agent in the bloodstream.

The inventors have also discovered that by transforming cells of ananimal, in vivo, it is possible to modulate the animal's immune system.In particular, by transforming cells of an animal, with a recombinantgene, by site-specific or systemic administration it is possible tomodulate the animal's immune system to sensitize the animal to themolecule for which the recombinant gene encodes. Alternatively, bytransforming cells of an animal with a recombinant gene, specifically ata site which determines the specificity of the immune system, such as,e.g., the thymus, it is possible to modulate the immune system of ananimal to suppress the immune response to the molecule encoded by therecombinant gene.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same become betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying figures, wherein:

FIGS. 1 and 2 illustrate the use of a catheter in accordance with theinvention to surgically or percutaneously implant cells in a bloodvessel or to transform in vivo cells present on the wall of a patient'sblood vessel;

FIG. 3 illustrates the relationship between the % of target cell lysisand the effector:target ratio for CTL cells; and

FIG. 4 illustrates the results of a Western blot analysis.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Thus, in one embodiment, the present invention is used to treatdiseases, such as inherited diseases, systemic diseases, diseases of thecardiovascular system, diseases of particular organs, or tumors byinstilling normal or transformed cells or by transforming cells.

The cells which may be instilled in the present method includeendothelium, smooth muscle, fibroblasts, monocytes, macrophages, andparenchymal cells. These cells may produce proteins which may have atherapeutic or diagnostic effect and which may be naturally occurring orarise from recombinant genetic material.

Referring now to the figures, wherein like reference numerals designateidentical or corresponding parts throughout the several views, and moreparticularly to FIG. 1 thereof, this figure illustrates the practice ofthe present invention with a catheter having a design as disclosed inU.S. Pat. No. 4,636,195, which is hereby incorporated by reference. Thiscatheter may be used to provide normal or genetically altered cells onthe walls of a vessel or to introduce vectors for the localtransformation of cells. In the figure, 5 is the wall of the bloodvessel. The figure shows the catheter body 4 held in place by theinflation of inflatable balloon means 1 and 2. The section of thecatheter body 4 situated between balloon means 1 and 2 is equipped withinstillation port means 3. The catheter may be further equipped with aguidewire means 6. FIG. 2 illustrates the use of a similar catheter,distinguished from the catheter illustrated in FIG. 1 by the fact thatit is equipped with only a single inflatable balloon means 2 and aplurality of instillation port means 3. This catheter may contain up totwelve individual instillation port means 3, with five beingillustrated.

In the case of delivery to an organ, the catheter may be introduced intothe major artery supplying the tissue. Cells containing recombinantgenes or vectors can be introduced through a central instillation portafter temporary occlusion of the arterial circulation. In this way,cells or vector DNA may be delivered to a large amount of parenchymaltissue distributed through the capillary circulation. Recombinant genescan also be introduced into the vasculature using the double ballooncatheter technique in the arterial circulation proximal to the targetorgan. In this way, the recombinant genes may be secreted directly intothe circulation which perfuse the involved tissue or may be synthesizeddirectly within the organ.

In one embodiment, the therapeutic agents are secreted by vascular cellssupplying specific organs affected by the disease. For example, ischemiccardiomyopathy may be treated by introducing angiogenic factors into thecoronary circulation. This approach may also be used for peripheral,vascular or cerebrovascular diseases where angiogenic factors mayimprove circulation to the brain or other tissues. Diabetes mellitus maybe treated by introduction of glucose-responsive insulin secreting cellsin the portal circulation where the liver normally sees a higher insulinconcentration than other tissues.

In addition to providing local concentrations of therapeutic agents, thepresent method may also be used for delivery of recombinant genes toparenchymal tissues, because high concentrations of viral vector andother vectors can be delivered to a specific circulation. Using thisapproach, deficiencies of organ-specific proteins may also be treated.For example, in the liver, α-antitrypsin inhibitor deficiency orhyperchloresterolemia may be treated by introduction of α-antitrypsin orthe LDL receptor gene. In addition, this approach may be used for thetreatment of a malignancy. Secretion of specific recombinant toxin genesinto the circulation of inoperable-tumors provides a therapeutic effect.Examples include acoustic neuromas or certain hemangiomas which areotherwise unresurrectable.

In clinical settings, these therapeutic recombinant genes are introducedin cells supplying the circulation of the involved organ. Although thearterial and capillary circulations are the preferred locations forintroduction of these cells, venous systems are also suitable.

In its application to the treatment of local vascular damage the presentinvention provides for the expression of proteins which ameliorate thiscondition in situ. In one embodiment, because vascular cells are foundat these sites, they are used as carriers to convey the therapeuticagents.

The invention thus, in one of its aspects, relies on genetic alterationof endothelial and other vascular cells or somatic cell gene therapy,for transmitting therapeutic agents (i.e., proteins, growth factors) tothe localized region of vessel injury. To successfully use genetransplantation in the cells, four requirements must be fulfilled.First, the gene which is to be implanted into the cell must beidentified and isolated. Second, the gene to be expressed must be clonedand available for genetic manipulation. Third, the gene must beintroduced into the cell in a form that will be expressed or functional.Fourth, the genetically altered cells must be situated in the vascularregion where it is needed.

In accordance with the present invention the altered cells orappropriate vector may be surgically, percutaneously, or intravenouslyintroduced and attached to a section of a patient's vessel wall.Alternatively, some of the cells existing on the patient's vessel wallare transformed with the desired genetic material or by directlyapplying the vector. In some instances, vascular cells which are notgenetically modified can be introduced by these methods to replace cellslost or damaged on the vessel surface.

Any blood vessel may be treated in accordance with this invention; thatis, arteries, veins, and capillaries. These blood vessels may be in ornear any organ in the human, or mammalian, body.

Introduction of normal or genetically altered cells into a blood vessel

This embodiment of the invention may be illustrated as follows:

I. Establishment of endothelial or other vascular cells in tissueculture.

Initially, a cell line is established and stored in liquid nitrogen.Prior to cryopreservation, an aliquot is taken for infection ortransfection with a vector, viral or otherwise, containing the desiredgenetic material.

Endothelial or other vascular cells may be derived enzymatically from asegment of a blood vessel, using techniques previously described in J.W. Ford, et al., In Vitro, 17, 40 (1981). The vessel is excised,inverted over a stainless steel rod and incubated in 0.1% trypsin inCa⁺⁺ - and Mg⁺⁺ - free Hank's balanced salt solution (BSS) with 0.125%EDTA at pH 8 for 10 min at 37° C.

Cells (0.4 to 1.5×10⁶) are collected by centrifugation and resuspendedin medium 199 (GIBCO) containing 10% fetal bovine serum, endothelialcell growth supplement (ECGS, Collaborative Research, Waltham, Mass.) at25 μg/ml, heparin at 15 U/ml, and gentamicin (50 μg/ml). Cells are addedto a 75 cm² tissue culture flask precoated with gelatin (2 mg/ml indistilled water). Cells are fed every second day in the above mediumuntil they reach confluence.

After two weeks in culture, the ECGS and heparin may be omitted from themedium when culturing porcine endothelium. If vascular smooth musclecells or fibroblasts are desired the heparin and ECGS can be omittedentirely from the culturing procedure. Aliquots of cells are stored inliquid nitrogen by resuspending to approximately 10⁶ cells in 0.5 ml ofice cold fetal calf serum on ice. An equal volume of ice cold fetal calfserum containing 10% DMSO is added, and cells are transferred to aprechilled screw cap Corning freezing tube. These cells are transferredto a -70° C. freezer for 3 hours before long term storage in liquidnitrogen.

The cells are then infected with a vector containing the desired geneticmaterial.

II. Introduction of cells expressing normal or exogenous proteins intothe vasculature.

A. Introduction of cells expressing relevant proteins bycatheterization.

The patient is prepared for catheterization either by surgery orpercutaneously, observing strict adherence to sterile techniques. Acutdown procedure is performed over the target blood vessel or a needleis inserted into the target blood vessel after appropriate anesthesia.The vessel (5) is punctured and a catheter, such as described in U.S.Pat. No. 4,636,195, which is hereby incorporated by reference (availablefrom USCI, Billerica, Mass.) is advanced by guidewire means (6) underfluoroscopic guidance, if necessary, into the vessel (5) (FIG. 1). Thiscatheter means (4) is designed to introduce infected endothelial cellsinto a discrete region of the artery. The catheter has a proximal anddistal balloon means (2) and (1), respectively, (e.g., each balloonmeans may be about 3 mm in length and about 4 mm in width), with alength of catheter means between the balloons. The length of cathetermeans between the balloons has a port means connected to an instillationport means (3). When the proximal and distal balloons are inflated, acentral space is created in the vessel, allowing for instillation ofinfected cells though the port.

A region of the blood vessel is identified by anatomical landmarks andthe proximal balloon means (2) is inflated to denude the endothelium bymechanical trauma (e.g., by forceful passage of a partially inflatedballoon catheter within the vessel) or by mechanical trauma incombination with small amounts of a proteolytic enzyme such as dispase,trypsin, collagenase, papain, pepsin, chymotrypsin or cathepsin, or byincubation with these proteolytic enzymes alone. In addition toproteolytic enzymes, liposes may be used. The region of the blood vesselmay also be denuded by treatment with a mild detergent or the like, suchas NP-40, Triton X100, deoxycholate, or SDS.

The denudation conditions are adjusted to achieve essentially completeloss of endothelium for cell transfers or approximately 20 to 90%,preferably 50 to 75%, loss of cells from the vessel wall for directinfection. In some instances cell removal may not be necessary. Thecatheter is then advanced so that the instillation port means (3) isplaced in the region of denuded endothelium. Infected, transfected ornormal cells are then instilled into the discrete section of artery overthirty minutes. If the blood vessel is perfusing an organ which cantolerate some ischemia, e.g., skeletal muscle, distal perfusion is not amajor problem, but can be restored by an external shunt if necessary, orby using a catheter which allows distal perfusion. After instillation ofthe infected endothelial cells, the balloon catheter is removed, and thearterial puncture site and local skin incision are repaired. If distalperfusion is necessary, an alternative catheter designed to allow distalperfusion may be used.

B. Introduction of recombinant genes directly into cells on the wall ofa blood vessel or perfused by a specific circulation in vivo; infectionor transfection of cells on the vessel wall and organs.

Surgical techniques are used as described above. Instead of usinginfected cells, a high titer desired genetic material transducing viralvector (10⁵ to 10⁶ particles/ml) or DNA complexed to a delivery vectoris directly instilled into the vessel wall using the double ballooncatheter technique. This vector is instilled in medium containing serumand polybrene (10 μg/ml) to enhance the efficiency of infection. Afterincubation in the dead space created by the catheter for an adequateperiod of time (0.2 to 2 hours or greater), this medium is evacuated,gently washed with phosphate-buffered saline, and arterial circulationis restored. Similar protocols are used for post operative recovery.

The vessel surface can be prepared by mechanical denudation alone, incombination with small amounts of proteolytic enzymes such as dispase,trypsin, collagenase or cathepsin, or by incubation with theseproteolytic enzymes alone. The denudation conditions are adjusted toachieve the appropriate loss of cells from the vessel wall.

Viral vector or DNA-vector complex is instilled in Dulbecco's modifiedEagle's medium using purified virus or complexes containing autologousserum, and adhesive molecules such as polybrene (10 μg/ml),poly-L-lysine, dextran sulfate, or any polycationic substance which isphysiologically suitable, or a hybrid antibody directed against theenvelope glycoprotein of the virus or the vector and the relevant targetin the vessel wall or in the tissue perfused by the vessel to enhancethe efficiency of infection by increasing adhesion of viral particles tothe relevant target cells. The hybrid antibody directed against theenvelope glycoprotein of the virus or the vector and the relevant targetcell can be made by one of two methods. Antibodies directed againstdifferent epitopes can be chemically crosslinked (G. Jung, C. J. Honsik,R. A. Reisfeld, and H. J. Muller-Eberhard, Proc. Natl. Acad. Sci. USA,83, 4479 (1986); U. D. Staerz, O. Kanagawa, and M. J. Bevan, Nature,314, 628 (1985); and P. Perez, R. W. Hoffman, J. A. Titus, and D. M.Segal, J. Exp. Med., 163, 166 (1986)) or biologically coupled usinghybrid hybridomas (U. D. Staerz and M. J. Bevan, Proc. Natl. Acad. Sci.USA, 83, 1453 (1986); and C. Milstein and A. C. Cuello, Nature, 305, 537(1983)). After incubation in the central space of the catheter for 0.2to 2 hours or more, the medium is evacuated, gently washed withphosphate buffered saline, and circulation restored.

Using a different catheter design (see FIG. 2), a different protocol forinstillation can also be used. This second approach involves the use ofa single balloon means (2) catheter with multiple port means (3) whichallow for high pressure delivery of the retrovirus into partiallydenuded arterial segments. The vessel surface is prepared as describedabove and defective vector is introduced using similar adhesivemolecules. In this instance, the use of a high pressure delivery systemserves to optimize the interaction of vectors with cells in adjacentvascular tissue.

The present invention also provides for the use of growth factorsdelivered locally by catheter or systemically to enhance the efficiencyof infection. In addition to retroviral vectors, herpes virus,adenovirus, or other viral vectors are suitable vectors for the presenttechnique.

It is also possible to transform cells within an organ or tissue. Directtransformation of organ or tissue cells may be accomplished by one oftwo methods. In a first method a high pressure transfection is used. Thehigh pressure will cause the vector to migrate through the blood vesselwalls into the surrounding tissue. In a second method, injection into acapillary bed, optionally after injury to allow leaking, gives rise todirect infection of the surrounding tissues.

The time required for the instillation of the vectors or cells willdepend on the particular aspect of the invention being employed. Thus,for instilling cells or vectors in a blood vessel a suitable time wouldbe from 0.01 to 12 hrs, preferably 0.1 to 6 hrs, most preferably 0.2 to2 hrs. Alternatively for high pressure instillation of vectors or cells,shorter times might be preferred.

Obtaining the cells used in this invention

The term "genetic material" generally refers to DNA which codes for aprotein. This term also encompasses RNA when used with an RNA virus orother vector based on RNA.

Transformation is the process by which cells have incorporated anexogenous gene by direct infection, transfection or other means ofuptake.

The term "vector" is well understood and is synonymous with theoften-used phrase "cloning vehicle". A vector is non-chromosomaldouble-stranded DNA comprising an intact replicon such that the vectoris replicated when placed within a unicellular organism, for example bya process of transformation. Viral vectors include retroviruses,adenoviruses, herpesvirus, papovirus, or otherwise modified naturallyoccurring viruses. Vector also means a formulation of DNA with achemical or substance which allows uptake by cells.

In another embodiment the present invention provides for inhibiting theexpression of a gene. Four approaches may be utilized to accomplish thisgoal. These include the use of antisense agents, either syntheticoligonucleotides which are complementary to the mRNA (Maher III, L. J.and Dolnick, B. J. Arch. Biochem. Biophys., 253, 214-220 (1987) andZamecnik, P. C., et al., Proc. Natl. Acad. Sci., 83, 4143-4146 (1986)),or the use of plasmids expressing the reverse complement of this gene(Izant, J. H. and Weintraub, H., Science, 229, 345-352, (1985); Cell,36, 1077-1015 (1984)). In addition, catalytic RNAs, called ribozymes,can specifically degrade RNA sequences (Uhlenbeck, O. C., Nature, 328,596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature, 334, 585-591(1988)). The third approach involves "intracellular immunization", whereanalogues of intracellular proteins can interfere specifically withtheir function (Friedman, A. D., Triezenberg, S. J. and McKnight, S. L.,Nature, 335, 452-454 (1988)), described in detail below.

The first approaches may be used to specifically eliminate transcriptsin cells. The loss of transcript may be confirmed by S1 nucleaseanalysis, and expression of binding protein determined using afunctional assay. Single-stranded oligonucleotide analogues may be usedto interfere with the processing or translation of the transcriptionfactor mRNA. Briefly, synthetic oligonucleotides or thiol-derivativeanalogues (20-50 nucleotides) complementary to the coding strand of thetarget gene may be prepared. These antisense agents may be preparedagainst different regions of the mRNA. They are complementary to the 5'untranslated region, the translational initiation site and subsequent20-50 base pairs, the central coding region, or the 3' untranslatedregion of the gene. The antisense agents may be incubated with cellstransfected prior to activation. The efficacy of antisense competitorsdirected at different portions of the messenger RNA may be compared todetermine whether specific regions may be more effective in preventingthe expression of these genes.

RNA can also function in an autocatalytic fashion to cause autolysis orto specifically degrade complementary RNA sequences (Uhlenbeck, O. C.,Nature, 328, 596-600 (1987), Haseloff, J. and Gerlach, W. L., Nature,334, 585-591 (1988), and Hutchins, C. J., et al, Nucleic Acids Res., 14,3627-3640 (1986)). The requirements for a successful RNA cleavageinclude a hammerhead structure with conserved RNA sequence at the regionflanking this structure. Regions adjacent to this catalytic domain aremade complementary to a specific RNA, thus targeting the ribozyme tospecific cellular mRNAs. To inhibit the production of a specific targetgene, the mRNA encoding this gene may be specifically degraded usingribozymes. Briefly, any GUG sequence within the RNA transcript can serveas a target for degradation by the ribozyme. These may be identified byDNA sequence analysis and GUG sites spanning the RNA transcript may beused for specific degradation. Sites in the 5' untranslated region, inthe coding region, and in the 3' untranslated region may be targeted todetermine whether one region is more efficient in degrading thistranscript. Synthetic oligonucleotides encoding 20 base pairs ofcomplementary sequence upstream of the GUG site, the hammerheadstructure and ˜20 base pairs of complementary sequence downstream ofthis site may be inserted at the relevant site in the cDNA. In this way,the ribozyme may be targeted to the same cellular compartment as theendogenous message. The ribozymes inserted downstream of specificenhancers, which give high level expression in specific cells may alsobe generated. These plasmids may be introduced into relevant targetcells using electroporation and cotransfection with a neomycin resistantplasmid, pSV2-Neo or another selectable marker. The expression of thesetranscripts may be confirmed by Northern blot and S1 nuclease analysis.When confirmed, the expression of mRNA may be evaluated by S1 nucleaseprotection to determine whether expression of these transcripts reducessteady state levels of the target mRNA and the genes which it regulates.The level of protein may also be examined.

Genes may also be inhibited by preparing mutant transcripts lackingdomains required for activation. Briefly, after the domain has beenidentified, a mutant form which is incapable of stimulating function issynthesized. This truncated gene product may be inserted downstream ofthe SV-40 enhancer in a plasmid containing the neomycin resistance gene(Mulligan, R. and Berg, P., Science, 209, 1422-1427 (1980) (in aseparate transcription unit). This plasmid may be introduced into cellsand selected using G418. The presence of the mutant form of this genewill be confirmed by S1 nuclease analysis and by immunoprecipitation.The function of the endogenous protein in these cells may be evaluatedin two ways. First, the expression of the normal gene may be examined.Second, the known function of these proteins may be evaluated. In theevent that this mutant intercellular interfering form is toxic to itshost cell, it may be introduced on an inducible control element, such asmetallothionein promoter. After the isolation of stable lines, cells maybe incubated with Zn or Cd to express this gene. Its effect on hostcells can then be evaluated.

Another approach to the inactivation of specific genes is to overexpressrecombinant proteins which antagonize the expression or function ofother activities. For example, if one wished to decrease expression ofTPA (e.g., in a clinical setting of disseminate thrombolysis), one couldoverexpress plasminogen activator inhibitor.

Advances in biochemistry and molecular biology in recent years have ledto the construction of "recombinant" vectors in which, for example,retroviruses and plasmids are made to contain exogenous RNA or DNA,respectively. In particular instances the recombinant vector can includeheterologous RNA or DNA, by which is meant RNA or DNA that codes for apolypeptide ordinarily not produced by the organism susceptible totransformation by the recombinant vector. The production of recombinantRNA and DNA vectors is well understood and need not be described indetail. However, a brief description of this process is included herefor reference.

For example, a retrovirus or a plasmid vector can be cleaved to providelinear RNA or DNA having ligatable termini. These termini are bound toexogenous RNA or DNA having complementary like ligatable termini toprovide a biologically functional recombinant RNA or DNA molecule havingan intact replicon and a desired phenotypical property.

A variety of techniques are available for RNA and DNA recombination inwhich adjoining ends of separate RNA or DNA fragments are tailored tofacilitate ligation.

The exogenous, i.e., donor, RNA or DNA used in the present invention isobtained from suitable cells. The vector is constructed using knowntechniques to obtain a transformed cell capable of in vivo expression ofthe therapeutic agent protein. The transformed cell is obtained bycontacting a target cell with a RNA- or DNA-containing formulationpermitting transfer and uptake of the RNA or DNA into the target cell.Such formulations include, for example, retroviruses, plasmids,liposomal formulations, or plasmids complexes with polycationicsubstances such as poly-L-lysine, DEAC-dextran and targeting ligands.

The present invention thus provides for the genetic alteration of cellsas a method to transmit therapeutic or diagnostic agents to localizedregions of the blood vessel for local or systemic purposes. The range ofrecombinant proteins which may be expressed in these cells is broad andvaried. It includes gene transfer using vectors expressing such proteinsas tPA for the treatment of thrombosis and restenosis, angiogenesis orgrowth factors for the purpose of revascularization, and vasoactivefactors to alleviate vasoconstriction or vasospasm. This technique canalso be extended to genetic treatment of inherited disorders, oracquired diseases, localized or systemic. The present invention may alsobe used to introduce normal cells to specific sites of cell loss, forexample, to replace endothelium damaged during angioplasty orcatheterization.

For example, in the treatment of ischemic diseases (thromboticdiseases), genetic material coding for tPA or modifications thereof,urokinase or streptokinase is used to transform the cells. In thetreatment of ischemic organ (e.g., heart, kidney, bowel, liver, etc.)failure, genetic material coding for recollateralization agents, such astransforming growth factor α (TGF-α), transforming growth factor β(TGF-β) angiogenin, tumor necrosis factor α, tumor necrosis factor β,acidic fibroblast growth factor or basic fibroblast growth factor can beused. In the treatment of vasomotor diseases, genetic material codingfor vasodilators or vasoconstrictors may be used. These include atrialnatriuretic factor, platelet-derived growth factor or endothelin. In thetreatment of diabetes, genetic material coding for insulin may be used.

The present invention can also be used in the treatment of malignanciesby placing the transformed cells in proximity to the malignancy. In thisapplication, genetic material coding for diphtheria toxin, pertussistoxin, or cholera toxin may be used.

In one of its embodiments, the present invention provides for thetherapy of malignancy by either stimulating an immune response againsttumor cells or inhibiting tumor cell growth or metastasis by geneticmodification in vivo. This approach differs from previous methods inwhich tumor cells are propagated, modified, and selected in vitro.

In accordance with this embodiment, the present method is used todeliver a DNA sequence or an RNA sequence, including recombinant genes,to tumor cells in vivo with (1) retroviral or viral vectors as vehicles,(2) DNA or RNA/liposome complexes as vehicles, (3) chemical formulationscontaining the DNA or RNA sequence and coupled to a carrier moleculewhich facilitates delivery of the sequence to the targeted cells, or (4)by utilizing cell-mediated gene transfer to deliver genes to specificsites in vivo, e.g., by relying upon the use of vascular smooth musclecells or endothelia cells which have been transduced in vitro as avehicle to deliver the recombinant gene into the site of the tumor.

In an aspect of this embodiment, the present invention relies on theimmune system to provide protection against cancer and play an importantrole as an adjuvant treatment for a malignancy. Immunotherapy has shownpromise as an adjuvant approach to the treatment of malignancies. Bothcytolytic T cells and lymphokines can facilitate tumor cell destruction,and strategies to enhance tumor regression by administration ofcytokines or tumor infiltrating lymphocytes have shown efficacy inanimal models and human trials. For example, it is known that lymphokineactivated killer cells (LAK) and tumor infiltrating lymphocytes (TIL)can lyse neoplastic cells and produce partial or complete tumorrejection. Expression of cytokine genes in malignant cells has alsoenhanced tumor regression.

The present invention provides a novel gene transfer approach againsttumors by the introduction of recombinant genes directly into tumorcells in vivo, where, by contrast, traditional gene transfer techniqueshave focused on modification of tumor cells in vitro followed bytransfer of the modified cells. The prior art approaches aredisadvantageous because they subject the cells to selection in differentgrowth conditions from those which act in vivo, and because they alsorequire that cell lines be established for each malignancy, therebyrendering adaptability to human disease considerably more difficult.

Genes which may be used with this embodiment include genes containing aDNA sequence (or the corresponding RNA sequence may be used) encoding anintracellular, secreted, or cell surface molecule which is exogenous tothe patient and which (1) is immunogenic to the patient, (2) inducesrejection, regression, or both, of the tumor, or (3) is toxic to thecells of the tumor.

The vectors containing the DNA sequence (or the corresponding RNAsequence) which may be used in accordance with the invention may be aneukaryotic expression vector containing the DNA or the RNA sequence ofinterest. Techniques for obtaining expression of exogenous DNA or RNAsequences in a host are known. See, for example, Korman et al, Proc.Nat. Acad. Sci. (USA), (1987) 84:2150-2154, which is hereby incorporatedby reference.

This vector, as noted above, may be administered to the patient in aretroviral or other viral vector (i.e., a viral vector) vehicle, a DNAor RNA/liposome complex, or by utilizing cell-mediated gene transfer.Further, the vector, when present in non-viral form, may be administeredas a DNA or RNA sequence-containing chemical formulation coupled to acarrier molecule which facilitates delivery to the host cell. Suchcarrier molecule would include an antibody specific to the cells towhich the vector is being delivered or a molecule capable of interactingwith a receptor associated with the target cells.

Cell-mediated gene transfer may be used in accordance with theinvention. In this mode, one relies upon the delivery of recombinantgenes into living organisms by transfer of the genetic material intocells derived from the host and modification in cell culture, followedby the introduction of genetically altered cells into the host. Anillustrative packaging cell line which may be used in accordance withthis embodiment is described in Danos et al, Proc. Natl. Acad. Sci.(USA) (1988) 85:6460, which is hereby incorporated by reference.

The DNA or RNA sequence encoding the molecule used in accordance withthe invention may be administered to the patient, which may be human ora non-human animal, either locally or systemically. The systemicadministration is preferably carried out using the non-viral DNA or RNAchemical formulation coupled to a carrier molecule which facilitatesdelivery to the host cells. Any of the administrations may be performedby IV or IM injection or subcutaneous injection using any known means,or by the use of the catheter in accordance with the present invention.

The retroviral vector vehicles used in accordance with the presentinvention comprise a viral particle derived from a naturally-occurringretrovirus which has been genetically altered to render it replicationdefective and to express a recombinant gene of interest in accordancewith the invention. Once the virus delivers its genetic material to acell, it does not generate additional infectious virus but doesintroduce exogenous recombinant genes to the cell.

In other viral vectors, the virus particle used is derived from othernaturally-occurring viruses which have been genetically altered torender them replication defective and to express recombinant genes. Suchviral vectors may be derived from adenovirus, papillomavirus,herpesvirus, parvovirus, etc.

The sequences of the present invention may also be administered as DNAor RNA/liposome complex. Such complexes comprise a mixture of fatparticles, lipids, which bind to genetic material, DNA or RNA, providinga hydrophobic coat, allowing genetic material to be delivered intocells. This formulation provides a non-viral vector for gene transfer.Liposomes used in accordance with the invention may comprise DOPE(dioleyl phosphatidyl ethanol amine), CUDMEDA (N-(5-cholestrum-3-β-ol3-urethanyl)-N',N'-dimethylethylene diamine).

As noted above, other non-viral vectors may also be used in accordancewith the present invention. These include chemical formulations of DNAor RNA coupled to a carrier molecule (e.g., an antibody or a receptorligand) which facilitates delivery to host cells for the purpose ofaltering the biologic properties of the host cells. The term "chemicalformulations" used herein refers to modifications of nucleic acids toallow coupling of the nucleic acid compounds to a protein or lipid, orderivative thereof, carrier molecule. Such carrier molecules includeantibodies specific to the host cells or receptor ligands, i.e.,molecules able to interact with receptors associated with the hostcells.

The molecules which may be used in accordance with this invention,include the following: (1) genes encoding immune stimulants, such asClass I histocompatibility genes, Class II histocompatibility genes,bacterial genes, including mycobacterial (PPD) genes and genes encodingheat shock proteins, viral glycoproteins encoding genes, includingvesicular stomatitis virus G protein, influenza hemagglutinin, andherpes virus glycoprotein β, minor histocompatibility antigens, foreignproteins, such as lysozyme or bovine serum albumin, and oncogenes,including EIA, P53 (mutants) and tax; (2) immune and growthstimulants/inhibitors, including inducers of differentiation, such asstimulants, including interleukin-2 (IL-2) IL-4, 3, 6 or 8,inhibitors/inducers of differentiation, such as TNF-α or β, TGF-β (1, 2or 3), IL-1, soluble growth factor receptors (PDGF, FGF receptors),recombinant antibodies to growth factors or receptors, analogs of growthfactors (PDGF, FGF), interferons (α, β or γ) and adhesion molecules; or(3) toxins or negative selectable markers, including thymidine kinase,diphtheria toxin, pertussis toxin or drug-sensitive proteins.

The DNA/RNA sequence is preferably obtained from a source of the samespecies as the patient, but this is not absolutely required, and thepresent invention provides for the use of DNA sequences obtained from asource of a species different from the patient in accordance with thisembodiment. A preferred embodiment of the present invention, genesencoding immune stimulants and toxins or negative selectable markers,corresponding to (1) and (3) above, are preferably selected from aspecies different than the species to which the patient belongs. Forimmune and growth stimulants/inhibitors, corresponding to (2) above, inaccordance with another preferred embodiment of the invention, onepreferably employs a gene obtained from a species which is the same asthe species of the patient.

In the use of the present invention in the treatment of AIDS, geneticmaterial coding for soluble CD4 or derivatives thereof may be used. Inthe treatment of genetic diseases, for example, growth hormonedeficiency, genetic material coding for the needed substance, forexample, human growth hormone, is used. All of these genetic materialsare readily available to one skilled in this art.

In another embodiment, the present invention provides a kit for treatinga disease in a patient which contains a catheter and a solution whichcontains either an enzyme or a mild detergent, in which the catheter isadapted for insertion into a blood vessel and contains a main catheterbody having a balloon element adapted to be inserted into said vesseland expansible against the walls of the blood vessel so as to hold themain catheter body in place in the blood vessel, and means carried bythe main catheter body for delivering a solution into the blood vessel,and the solution which contains the enzyme or mild detergent is aphysiologically acceptable solution. The solution may contain aproteolytic enzyme, such as dispase, trypsin, collagenase, papain,pepsin, or chymotrypsin. In addition to proteolytic enzymes, liposes maybe used. As a mild detergent, the solution may contain NP-40, TritonX100, deoxycholate, SDS or the like.

Alternatively, the kit may contain a physiological acceptable solutionwhich contains an agent such as heparin, poly-L-lysine, polybrene,dextran sulfate, a polycationic material, or bivalent antibodies. Thissolution may also contain vectors or cells (normal or transformed). Inyet another embodiment the kit may contain a catheter and both asolution which contains an enzyme or mild detergent and a solution whichcontains an agent such as heparin, poly-L-lysine, polybrene, dextransulfate, a polycationic material or bivalent antibody and which mayoptionally contain vectors or cells.

The kit may contain a catheter with a single balloon and central distalperfusion port, together with acceptable solutions to allow introductionof cells in a specific organ or vectors into a capillary bed or cells ina specific organ or tissue perfused by this capillary bed.

Alternatively, the kit may contain a main catheter body which has twospaced balloon elements adapted to be inserted in a blood vessel withboth being expansible against the walls of the blood vessel forproviding a chamber in the blood vessel, and to hold the main catheterbody in place. In this case, the means for delivering a solution intothe chamber is situated in between the balloon elements. The kit maycontain a catheter which possesses a plurality of port means fordelivering the solution into the blood vessel.

Thus, the present invention represents a method for treating a diseasein a patient by causing a cell attached onto the walls of a vessel orthe cells of an organ perfused by this vessel in the patient to expressan exogenous therapeutic agent protein, wherein the protein treats thedisease or may be useful for diagnostic purposes. The present method maybe used to treat diseases, such as an ischemic disease, a vasomotordisease, diabetes, a malignancy, AIDS or a genetic disease.

The present method may use exogenous therapeutic agent proteins, such astPA and modifications thereof, urokinase, streptokinase, acidicfibroblast growth factor, basic fibroblast growth factor, tumor necrosisfactor α, tumor necrosis factor β, transforming growth factor α,transforming growth factor β, atrial natriuretic factor,platelet-derived growth factor, endothelian, insulin, diphtheria toxin,pertussis toxin, cholera toxin, soluble CD4 and derivatives thereof, andgrowth hormone to treat diseases.

The present method may also use exogenous proteins of diagnostic value.For example, a marker protein, such as β-galactosidase, may be used tomonitor cell migration.

It is preferred, that the cells caused to express the exogenoustherapeutic agent protein be endothelial cells.

Other features of the present invention will become apparent in thecourse of the following descriptions of exemplary embodiments which aregiven for illustration of the invention and are not intended to belimiting thereof.

The data reported below demonstrate the feasibility of endothelial celltransfer and gene transplantation; that endothelial cells may be stablyimplanted in situ on the arterial wall by catheterization and express arecombinant marker protein, β-galactosidase, in vivo.

Because atherogenesis in swine has similarities to humans, an inbred pigstrain, the Yucatan minipig (Charles River Laboratories, Inc.,Wilmington, Mass.), was chosen as an animal model (1). A primaryendothelial cell line was established from the internal jugular vein ofan 8 month-old female minipig. The endothelial cell identity of thisline was confirmed in that the cells exhibited growth characteristicsand morphology typical of porcine endothelium in tissue culture.Endothelial cells also express receptors for the acetylated form of lowdensity lipoprotein (AcLDL), in contrast to fibroblasts and othermesenchymal cells (2). When analyzed for ACLDL receptor expression,greater than 99% of the cultured cells contained this receptor, asjudged by fluorescent ACLDL uptake.

Two independent β-galactosidase-expressing endothelial lines wereisolated following infection with a murine amphotropicβ-galactosidase-transducing retroviral vector (BAG), which isreplication-defective and contains both β-galactosidase and neomycinresistance genes (3). Cells containing this vector were selected fortheir ability to grow in the presence of G-418. Greater than 90% ofselected cells synthesized β-galactosidase by histochemical staining.The endothelial nature of these genetically altered cells was alsoconfirmed by analysis of fluorescent ACLDL uptake. Infection by BAGretrovirus was further verified by Southern blot analysis which revealedthe presence of intact proviral DNA at approximately one copy pergenome.

Endothelial cells derived from this inbred strain, being syngeneic, wereapplicable for study in more than one minipig, and were tested in ninedifferent experimental subjects. Under general anesthesia, the femoraland iliac arteries were exposed, and a catheter was introduced into thevessel (FIG. 1). Intimal tissues of the arterial wall were denudedmechanically by forceful passage of a partially inflated ballooncatheter within the vessel. The artery was rinsed with heparinizedsaline and incubated with the neutral protease, dispase (50 U/ml), whichremoved any remaining luminal endothelial cells. Residual enzyme wasrapidly inactivated by α2 globulin in plasma upon deflating the catheterballoons and allowing blood to flow through the vessel segment. Thecultured endothelial cells which expressed β-galactosidase wereintroduced using a specially designed arterial catheter (USCI,Billerica, Mass.) that contained two balloons and a central instillationport (FIG. 1).

When these balloons were inflated, a protected space was created withinthe artery into which cells were instilled through the central port 3(FIG. 1). These endothelial cells, which expressed β-galactosidase, wereallowed to incubate for 30 minutes to facilitate their attachment to thedenuded vessel. The catheter was then removed, the arterial branchligated, and the incision closed.

Segments of the artery inoculated with β-galactosidase-expressingendothelium were removed 2 to 4 weeks later. Gross examination of thearterial specimen after staining using the X-gal chromogen showedmultiple areas of blue coloration, compared to an artery seeded withuninfected endothelium, indicative of β-galactosidase activity. Lightmicroscopy documented β-galactosidase staining primarily in endothelialcells of the intima in experimentally seeded vessels.

In contrast, no evidence of similar staining was observed in controlsegments which had received endothelial cells containing noβ-galactosidase. β-Galactosidase staining was occasionally evident indeeper intimal tissues, suggesting entrapment or migration of seededendothelium within the previously injured vessel wall. Local thrombosiswas observed in the first two experimental subjects. This complicationwas minimized in subsequent studies by administering acetylsalicylicacid prior to the endothelial cell transfer procedure and use of heparinanticoagulation at the time of inoculation. In instances of thrombusformation, β-galactosidase staining was seen in endothelial cellsextending from the vessel wall to the surface of the thrombus.

A major concern of gene transplantation in vivo relates to theproduction of replication-competent retrovirus from geneticallyengineered cells. In these tests, this potential problem has beenminimized through the use of a replication defective retrovirus. Nohelper virus was detectable among these lines after 20 passages invitro. Although defective viruses were used because of their high rateof infectivity and their stable integration into the host cell genome(4), this approach to gene transfer is adaptable to other viral vectors.

A second concern involves the longevity of expression of recombinantgenes in vivo. Endothelial cell expression of β-galactosidase appearedconstant in vessels examined up to six weeks after introduction into theblood vessel in the present study.

These tests have demonstrated that genetically-altered endothelial cellscan be introduced into the vascular wall of the Yucatan minipig byarterial catheterization. Thus, the present method can be used for thelocalized biochemical treatment of vascular disease usinggenetically-altered endothelium as a vector.

A major complication of current interventions for vascular disease, suchas balloon angioplasty or insertion of a graft into a diseased vessel,is disruption of the atherosclerotic plaque and thrombus formation atsites of local tissue trauma (5). In part, this is mediated byendothelial cell injury (6). The present data show thatgenetically-altered endothelial cells can be introduced at the time ofintervention to minimize local thrombosis.

This technique can also be used in other ischemic settings, includingunstable angina or myocardial infarction. For instance, antithromboticeffects can be achieved by introducing cells expressing genes for tissueplasminogen activator or urokinase. This technology is also useful forthe treatment of chronic tissue ischemia. For example, elaboration ofangiogenic or growth factors (7) to stimulate the formation ofcollateral vessels to severely ischemic tissue, such as the myocardium.Finally, somatic gene replacement for systemic inherited diseases isfeasible using modifications of this endothelial cell gene transfertechnique.

Another aspect of the present invention relates a method for modulatingthe immune system of an animal by in vivo transformation of cells of theanimal with a recombinant gene. The transformation may be carried outeither in a non-site-specific or systemic manner or a site-specificmanner. If the transformation is carried out in a systemic fashion or atsites other than those which confer specificity on the immune system,such as the thymus, then the immune system will be modulated to resultin the animal being sensitized to the molecule for which the recombinantgene encodes. Alternatively, if the transformation is carried out in asite-specific manner and is localized to a site which determines thespecificity of the immune system, e.g., the thymus, the immune systemwill be modulated to result in the animal being tolerized to themolecule encoded by the recombinant gene.

By the term sensitized, it is meant that the immune system exhibits astronger response to the molecule encoded by the DNA after in vivotransformation as compared to before transformation. By the termtolerized, it is meant that the immune system displays a reducedresponse to the molecule encoded by the recombinant gene aftertransformation as compared to before transformation. Thus, one maymodulate an immune system to provide either a resistance or a toleranceto the molecule encoded by the DNA.

Examples of molecules for which it may be desirable to provide aresistance to include: cell surface molecules, such as tumor antigens(carcinoembryonic antigen), protozoan antigens (pneumocystis), viralantigens (HIV gp120 and gp160, H. influenza antigen, and hepatitis Bsurface antigen), Lyme disease antigen, Bacterial antigens, andtransplantation antigens (Class I or II), ras or other oncogenes,including erb-A or neu; cytoplasmic proteins, such as the raf oncogene,src oncogene, and abl oncogene; nuclear proteins, such as E1A oncogene,mutant p53 oncogene, tat, tax, rev, vpu, vpx, hepatitis core antigen,EBNA and viral genes; and secreted proteins, such as endotoxin, choleratoxin, TNF, and osteoclast activating factor.

Examples of molecules for which it may be desirable to provide aresistance to include: cell surface molecules, such as growth factorreceptors, insulin receptors, thyroid hormone receptors, transplantationantigens (class I or II), blood group antigens, and LDL receptor;cytoplasmic proteins, such as cytochrome P450, galactosyl transferase,dystrophin, neomycin resistance gene, and bacterial heat shock protein;nuclear proteins, such as retinoblastoma and transdominant rev; andsecreted proteins, such as growth hormone for dwarfs, insulin fordiabetics, and adenosine deaminase.

It is to be understood that the nucleic acid, DNA, RNA, or derivativethereof, in the recombinant gene may be of any suitable origin. That isthe nucleic acid may be isolated from a naturally occurring source ormay be of synthetic origin.

The recombinant gene may be introduced in the cells of the animal usingany conventional vector. Such vectors include viral vectors, cationiclipids complexed to DNA or RNA (DNA or RNA/liposomes) and DNA or RNAcomplexes with polycations, such as DEAE, dextran, and polybrene.

As noted above the recombinant gene can be introduced into cells in asite-specific manner to confer resistance to the molecule encoded by therecombinant gene. Suitable sites include, e.g., endothelial cells orreticuloendothelial cells in the vasculature or any specific tissue ororgan. The form of the preparation containing the vector and recombinantgene used in the transformation will depend on the specific tissue to betransformed. Suitable preparations for transforming endothelial cellsare described elsewhere in this specification. In addition, preparationssuitable for oral or other means of administration (e.g., endoscopic)may be used to provide mucosal resistance. Such preparation couldinclude detergents, gelatins, capsules or other delivery vehicles toprotect against degradation and enhance delivery to the mucosal surface,in addition to the vector and gene.

Alternatively, the recombinant gene may be introduced in a site specificfashion to a site which determines the specificity of the immune system.The thymus is such a site (see: A. M. Posselt et al, Science, vol. 249,p. 1292 (1990)). Thus, by introducing a recombinant genesite-specifically into the thymus, the immune system may be modulated toresult in a tolerance to the molecule encoded by the gene. In this way,transplant rejection may be suppressed. The same preparations andtechniques used to site-specifically transform tumors described abovemay be used to introduce the recombinant gene into the thymus.Specifically, the transformation preparation may be injected directedinto the thymus or tumor or into the vascular supply of the thymus ortumor.

The present method may be practiced on any animal, such as chickens ormammals such as cows, horses, cats, dogs, monkeys, lemurs or humans.

When the recombinant gene is introduced using a liposome, it ispreferred to first determine in vitro the optimal values for the DNA:lipid ratios and the absolute concentrations of DNA and lipid as afunction of cell death and transformation efficiency for the particulartype of cell to be transformed and to use these values in the in vivotransformation. The in vitro determination of these values can be easilycarried out using the techniques described in the Experimental Sectionof this specification.

Another aspect of the present invention relates to a kit for the in vivosystemic introduction of a recombinant gene into cells of an animal.Such a kit would include approximately the optimal amount of a carrier,such as a lipid, and nucleic acid, and/or a means of delivery, e.g., anendoscope or a syringe. The kit may also contain instructions for theadministration of the transforming preparation. The carrier and nucleicacid may be freeze dried and may be packaged separately or premixed. Thekit may also contain a solution to optimally reconstitute the complexesof the carrier and the nucleic acid, which provide for efficientdelivery to cells in vivo. Such a solution may contain one or moreingredients, such as buffers, sugars, salts, proteins, and detergents.

Having generally described the invention, a further understanding can beobtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

Experimental section

A. Analysis of AcLDL receptor expression in normal andβ-galactosidase-transduced porcine endothelial cells.

Endothelial cell cultures derived from the Yucatan minipig, two sublinesinfected with BAG retrovirus or 3T3 fibroblast controls were analyzedfor expression of AcLDL receptor using fluorescent labelled AcLDL.

Endothelial cells were derived from external jugular veins using theneutral protease dispase (8). Excised vein segments were filled withdispase (50 U/ml in Hanks' balanced salt solution) and incubated at 30°C. for 20 minutes. Endothelium obtained by this means was maintained inmedium 199 (GIBCO, Grand Island, N.Y.) supplemented with fetal calfserum (10%), 50 μg/ml endothelial cell growth supplement (ECGS) andheparin (100 μg/ml). These cells were infected with BAG retrovirus, andselected for resistance to G-418. Cell cultures were incubated with(1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbacyanine perchlorate)(Dil) AcLDL (Biomedical Technologies, Stoughton, Mass.) (10 μg/ml) for4-6 hrs. at 37° C., followed by three rinses with phosphate-bufferedsaline containing 0.5% glutaraldehyde. Cells were visualized by phasecontrast and fluorescent microscopy.

B. Method of introduction of endothelial cells by catheterization.

A double balloon catheter was used for instillation of endothelialcells. The catheter has a proximal and distal balloon, each 6 mm inlength and 5 mm in width, with a 20 mm length between the balloons. Thecentral section of the catheter has a 2 mm pore connected to aninstillation port. Proximal and distal balloon inflation isolates acentral space, allowing for instillation of infected cells through theport into a discrete segment of the vessel. For a schematicrepresentation of cell introduction by catheter, see FIGS. 1 and 2.

Animal care was carried out in accordance with "Principles of LaboratoryAnimal Care" and "Guide for the Care and Use of Laboratory Animals" (NIHpublication No. 80-23, Revised 1978). Female Yucatan minipigs (80-100kg) were anesthetized with pentobarbital (20 mg/kg), intubated, andmechanically ventilated. These subjects underwent sterile surgicalexposure of the iliac and femoral arteries. The distal femoral arterywas punctured, and the double-balloon catheter was advanced by guidewireinto the iliac artery. The external iliac artery was identified; theproximal balloon was partially inflated and passed proximally anddistally so as to mechanically denude the endothelium. The catheter wasthen positioned with the central space located in the region of denudedendothelium, and both balloons were inflated. The denuded segment wasirrigated with heparinized saline, and residual adherent cells wereremoved by instillation of dispase (20 U/ml) for 10 min. The denudedvessel was further irrigated with a heparin solution and theBAG-infected endothelial cells were instilled for 30 min. The ballooncatheter was subsequently removed, and antegrade blood flow wasrestored. The vessel segments were excised 2 to 4 weeks later. A portionof the artery was placed in 0.5% glutaraldehyde for five minutes andstored in phosphate-buffered saline, and another portion was mounted ina paraffin block for sectioning. The presence of retroviral expressedβ-galactosidase was determined by a standard histochemical technique(19).

C. Analysis of endothelial cells in vitro and in vivo.

β-Galactosidase activity was documented by histochemical staining in (A)primary endothelial cells from the Yucatan minipig, (B) a sublinederived by infection with the BAG retroviral vector, (C) a segment ofnormal control artery, (D) a segment of artery instilled withendothelium infected with the BAG retroviral vector, (E) microscopiccross-section of normal control artery, and (F) microscopiccross-section of artery instilled with endothelium infected with the BAGretroviral vector.

Endothelial cells in tissue culture were fixed in 0.5% glutaraldehydeprior to histochemical staining. The enzymatic activity of the E. coliβ-galactosidase protein was used to identify infected endothelial cellsin vitro and in vivo. The β-galactosidase transducing Mo-MuLV vector(2), (BAG) was kindly provided by Dr. Constance Cepko. This vector usedthe wild type Mo-MuLV LTR as a promoter for the β-galactosidase gene.The simian virus 40 (SV-40) early promoter linked to the Tn5 neomycinresistance gene provides resistance to the drug G-418 and is inserteddownstream of the β-galactosidase gene, providing a marker to select forretrovirus-containing, β-galactosidase expressing cells. This defectiveretrovirus was prepared from fibroblast ψ am cells (3,10), andmaintained in Dulbecco's modified Eagle's medium (DMEM) and 10% calfserum. Cells were passaged twice weekly following trypsinization. Thesupernatant, with titers of 10⁴ -10⁵ /ml G-418 resistant colonies, wasadded to endothelial cells at two-thirds confluence and incubated for 12hours in DMEM with 10% calf serum at 37° C. in 5% CO₂ in the presence of8 μg/ml of polybrene. Viral supernatants were removed, and cellsmaintained in medium 199 with 10% fetal calf serum, ECGS (50 μg/ml), andendothelial cell conditioned medium (20%) for an additional 24 to 48hours prior to selection in G-418 (0.7 pg/ml of a 50% racemic mixture).G-418 resistant cells were isolated and analyzed for β-galactosidaseexpression using a standard histochemical stain (9). Cells stablyexpressing the β-galactosidase enzyme were maintained in continuousculture for use as needed. Frozen aliquots were stored in liquidnitrogen.

D. Immunotherapy of Malignancy by In Vivo Gene Transfer.

A retroviral vector which the H-2K^(S) gene was prepared. CT26 cellswere infected with this vector in vitro, selected for G418 resistance,and analyzed by fluorescence activated cell sorting (FACS). TransducedCT26 cells showed a higher mean fluorescence intensity than uninfectedCT26 cells or CT26 infected with different retroviral vectors. When 10⁶CT26 cells which express H-2K^(S) were injected subcutaneously intoBALB/c mice (H-2^(d)) sensitized to this antigen, no tumors wereobserved over an 8-week period in contrast to the unmodified CT26(H-2^(d)) tumor line which routinely formed tumors at this dose. Theimmune response to H-2K^(S) could therefore provide protection againstCT26 cells bearing this antigen. When CT26 H-2K^(S) and CT26 wereco-inoculated, however, tumor growth was observed, suggesting thatH-2K^(S) conferred senstivity only to modified cells.

To determine whether protective effects could be achieved byintroduction of H-2K^(S) in growing CT26 tumors, the recombinantH-2K^(S) reporter or a β-galactosidase gene was introduced into tumorseither with a DNA/liposome or a retroviral vector. Tumor capsules (0.5-1cm diameter) were exposed surgically and multiple needle injections(2-10) delivered to the parenchyma. With β-galactosidase reporterplasmids, recombinant gene expression could be readily detected afterintra-tumor injection of DNA/liposome or retroviral vectors.

In mice which received intra-tumor injections of the H-2K^(S)DNA/liposome comples or H-2K^(S) retroviral vector, the recombinant DNAwas detected by PCR in the tumor and occasioinally in other tissues.When found in the other organs, no evidence of inflammation or ograntoxicity was detected pathologically. An immune response to therecombinant H-2K^(S) protein was evident in these animals, however.Lymphocytes derived from the H-2K^(S), but not β-galactosidasetransduced tumors, demonstrated a cytolytic response to H-2K^(S) whetherdelivered by retroviral vectors or liposomes. More importantly,lymphocytes derived from the H-2K^(S), but not β-galactosidasetransduced animals, recognized and lysed unmodified CT26 cells,indicating that this stimulation induced immune reactivity againstgenetically unmodified tumor cells.

To assess the protective effect of the immune response against H-2K^(S),tumor growth in vivo was quantitated. When animals received no priorsensitization to H-2K^(S), one of four tumors transduced with H-2K^(S)showed attenuation of tumor growth which was not complete. In contrast,no anti-tumor effect was seen in unmodified (n=4) or β-galactosidasetransduced controls (n=4). Because these tumors were large at the timeof initial injection and continued to grow as the primary immuneresponse was generated, an attempt was made to optimize the anti-tumorresponse by pre-immunization of mice with irradiated CT26 H-2K^(S) tumorcells, and by earlier and/or more frequent injections of vector. Tumorswere transduced on days 12 and 32 by intra-tumor injuection of H-2K^(S)or β-galactosidase DNA/liposome vectors. Treatment with the H-2K^(S)liposome complex improved survival and attenuated tumor growth, incontrast to β-galactosidase transduced tumors where there was nodifference in growth rate compared to the uninjected controls. Completetumor regression was acheived in two mice by increasing the number ofinjections and by delivery of H-2K^(S) into tumors at an earlier stage.This treatment was protective, since control animals showed continuedtumor growth and did not survive beyond 35 days.

E. Modulation of the Immune System.

The response to injection of cationic lipids and plasmids was determinedafter injection intravenously into BALB/c mice (6-12 weeks). In thefirst experiments, a gene encoding the H-2K^(S) molecule was introducedby tail vein injection. Two to four weeks later, spleen cells wereharvested and analyzed for their ability to mediate a cytolytic T cellresponse. When these cells were tested using ⁵¹ Cr target cells (CT26cells expressing the H-2K^(S) gene), significant cytolysis was observedwhich was not seen in animals injected with the cointrol vector,β-galactosidase (see FIG. 3). Up to 25% of target cells were lysed ateffector: target ratios of 25:1.

In addition to this specific cytolytic T cell response, serologic orantibody responses to genes encoded by expression vector plasmids havebeen examined. When a plasmnid encoding the gp160 molecule of HIV isinjected, an antibody response is elicited in treated mice. In contrastto control animals injected with cationic lipids containingβ-galactosidase, mice injected with cationic lipids with gp 160 plasmidshowed an antibody response to the gp160 and gp120 form of this moleculeby Westerm blot analysis (See FIG. 4). These results demonstrate thatsystemic administration of cationic lipid/DNA complexes can be usedsuccessfully to induce cell-mediated and antibody-mediated immunityagainst foreign pathogens.

F. Determination of Optimal Transfection Conditions.

(1) Plasmid Construction

A plasmid containing the E. coli lacZ gene under the control of the RousSarcoma Virus LTS (RSV-β-gal) (Norton and Coffin, Mol. Cell. Biol.,5(2), 281-290, 1985) was used for transfection of porcine primaryendothelial and HeLa cells. In addition, a plasmid containing the lacZgene under the control of preproendothelin-1 5'-flanking DNA (-1410 to+83) (Wilson et al., Mol. Cell. Biol., 10(9), 4854-4862, 1990) was usedfor transfection of endothelial cells. For in vivo toxicity analysis,the RSV-β-gal plasmid, and a plasmid derived from the PLJ vectorcontaining the cDNA encoding an H-2K^(S) mouse MHC class I gene wereused.

(2) Cell Culture, Transfection Analysis, and Toxicity in Vitro

Primary endothelial cells, derived from the Yucatan minipig (YPE cells),were incubated with medium 199 (M199) supplemented with 10% FBS, 2 mM1-glutamine, 50 U/ml penicillin, and 5 μg/ml streptomycin. HeLa cellswere maintained in Dulbeccos Modified Eagles Medium (DMEM) supplementedwith 5% FBS, 2 mM 1-glutamine, 50 U/ml penicillin and 5 μg/mlstreptomycin. The DNA liposome mixture was prepared with lipidconcentrations of DOPE/DC-Chol between 2.5 and 25 μM added to 0.2 ml ofserum-free media or Ringer's lactate solution in polystyrene tubes.After mixing gently, the solution was allowed to stand at roomtemperature for 15-20 minutes. For transfection analysis, cells weregrown in 60 mm tissue culture dishes at 75% confluency or greater. Cellswere washed twice with serum-free media or lactated Ringers solution andthen placed in 0.5 mls of the same media. The DNA liposome solution (0.2ml) was then added slowly to the cells, with gentle mixing, with a finalvolume of 0.7 ml. This resulted in DNA concentrations between 0.7 and 7μg/ml (13-130 nM), and lipid concentrations of 7-70 μM. Transfection wasallowed to proceed for 1-5 hours, after which the cells were placed inmedia supplemented as decribed above. At 24-48 hours after transfectionthe enzymatic activity of the E. coli β-galactosidase protein was usedto identify transfected cells by staining with the X-gal chromagen.Toxicity in vitro was assessed by cytopathic effect or trypan blueexclusion.

(3) Animal Studies

For intravenous injections, the DNA/liposomes were prepared as describedfor the in vitro transfection studies in 0.2 ml of serum-free M199 orlactated-Ringers solution. After 15-20 min of incubation, the mixturewas diluted to 0.7 ml and 0.1 to 0.2 ml of this dilution was theninjected immediately into the tail vein of adult, female BALB/c mice.Blood was collected before injection and 9-11 days following injection,and serum chemistries were examined. At .sup.˜ 2-3 weeks followinginjection, the liver, kidney, lung, heart, and brain were extracted forhistologic and PCR DNA amplification analysis as described previously.Intratumor injection of CT26 cells (Fearon et al., Cell, 60, 397-403,1990) and analysis were also performed according to the previousprotocols.

(4) Results

The optimal conditions for transfection and toxicity of DNA/liposomeswere initially determined in vitro. To obtain maximal transfectionwithout toxicity in vitro, the ratio of DNA to cationic lipid, theabsolute concentration of DNA or lipids, and the conditions for mixtureof DNA and cationic lipids were studied. The cationic lipid preparationwas a formulation of two compounds, which include dioleoylphosphatidylethanolamine (DOPE) and cholesten-3-β-ol 3-urethanyl-N',N'dimethylethylene diamine (DC-chol). The transfection efficiencies ofthis reagent were equal to or greater than those of Lipofectin® (BRL) inseveral cell lines in vitro. Endothelial cells, which are typicallydifficult to transfect, and HeLa cells, which can be transfected easilyusing a variety of techniques, were examined by transfection in vitro.

To determine the optimal conditions for transfection of endothelialcells, the lipid was initially used at different concentrations whilethe DNA concentration was held constant. Maximal transfection efficiencywas seen using 0.7 μg/ml DNA (13 nM) and 21 μM of DOPE/DC0Chol lipid,with a sharp decline in the number of transfected cells with higher orlower lipid concentrations. Next, the DNA concentration was altered asthe lipid concentration remained constant. This analysis revealed asimilar sensitivity to DNA concentration, with the number of transfectedcells decreasing significantly with increments of DNA concentration aslow as 0.4 μg/ml. These results indicate that the ratio of DNA to lipidis important for maximum transfection efficiency, and that the absoluteconcentration of each component is also important in determining theefficiency of transfection. An incrase in DNA and lipid concentrationbeyond the optimal concentration of 0.7 μg/ml DNA (13 nM) and 21 μM ofDOPE/DC-Chol reduced the number of viable cells and did not increase thetransfection efficiency of the remaining viable cells. Lipidconcentrations greater than 35 μM reduced the number of viable cells by50% compared to the untransfected control, whereas the optimalconcentration of 0.7 μg/ml DNA (13 nM) ande 21 μM of lipid had no effecton cell viability after 5 hours of incubation.

To compare the optimal concentrations of transfection in a differentcell type, transfections were performed on HeLa cells. In this case, aslightly different optimal ratio of DNA and lipid were observed. Peaktransfection efficiencies were obtained at the same lipid concentrationas endothelial cells (21 μg/ml) but varied less with small differencesin DNA concentrations. DNA concentrations of 1.4-4.2 μg/ml were equallyeffective. Again, when the ratio of DNA to lipid was maintained but theconcentration of each was decreased three-fold, very few cells weretransfected, illustrating that both the ratio of DNA to lipid and theabsolute concentration of each component are important in maximizing thenumber of transfected cells. If HeLa cells were transfected at >80%confluence or greater, there was no toxicity using up to 35 μM of lipid.When cells were transfected at a lower saturation density, however, cellviability was reduced dramatically with as little as 7 μM of lipidcompared to the untransfected control cells. These results demonstratethat the optimal conditions for transfection and toxicity may differsomewhat depending on the cell line.

Another variable in the preparation of liposomes was the composition ofthe solution used to generate complexes of the cationic lipids with DNA.Among several media solutions analyzed, no substantial difference wasnoted in transfection efficiency or toxicity with M199, McCoys, OptiMEM,or RPMI media. A significant improvement in transfection efficiency wasobserved, however, using standard Ringers lactate. The number oftransfected cells increased more than 3-fold compared to the serum-freemedium, although prolonged incubation (≧2 hours) resulted in a loss ofcell viability in some cell types.

PUBLICATIONS CITED

1. J. S; Reitman, R. W. Mahley, D. L. Fry, Atherosclerosis 43, 119(1982).

2. R. E. Pitos, T. L. Innerarity, J. N. Weinstein, R. W. Mahley,Arteriosclerosis 1, 177 (1981); T. J. C. Van Berkel, J. F. Kruijt FEBSLett, 132, 61 (1981); J. C. Voyta, P. A. Netland, D. P. Via, E. P.Zetter, J. Cell. Biol., 99, 81A (abstr.) (1984); J. M. Wilson, D. E.Johnston, D. M. Jefferson, R. C. Mulligan, Proc. Natl. Acad. Sci.U.S.A., 84, 4421 (1988).

3. J. Price, D. Turner, C. Cepko, Proc. Natl. Acad. Sci. U.S.A., 84, 156(1987).

4. R. Mann, R. C. Mulligan, D. Baltimore, Cell 33, 153 (1983); C. L.Cepko, B. E. Floberts, R. C. Mulligan, Cell 37, 1053 (1984); M. A.Eglitis, W. F. Anderson, Biotechniques 6, 608 (1988).

5. S. G. Ellis, G. S. Roubin, S. B. King, J. S. Douglas, W. S. Weintraubet al., Circulation 77, 372 (1988); L. Schwartz, M. G. Bourassa, J.Lesperance, H. E. Aldrige, F. Kazim, et al., N. Engl. J. Med. 318, 1714(1988).

6. P. C. Block, R. K. Myler, S. Stertzer, J. T. Fallon, N. Engl. J. Med.305, 382 (1981); P. M. Steele, J. H. Chesebro, A. W. Stanson, Circ. Res.57, 105 (1985); J. R. Wilentz, T. A. Sanborn, C. C. Handenschild, C. R.Valeri, T. J. Ryan, D. P. Faxon, Circulation 75, 636, (1987); W.McBride, R. A. Lange, L. D. Hillis, N. Engl. J. Med. 318, 1734 (1988).

7. J. Folkman, M. Klagsbrun, Science 235, 442 (1987); S. J. Leibovich,P. J. Polverini, H. Michael Shepard, D. M. Wiseman, V. Shively, N.Nuseir, Nature 329, 630 (1987); J. Folkman, M. Klagsbrun, Nature 329,671 (1987).

8. T. Matsumura, T. Yamanka, S. Hashizume, Y. Irie, K. Nitta, Japan. J.Exp. Med. 45, 377 (1975); D. G. S. Thilo, S. Muller-Kusel, D. Heinrich,I. Kauffer, E. Weiss, Artery, 8, 25a (1980).

9. A. M. Dannenberg, M. Suga, in Methods for Studying MononuclearPhagocytes, D. O. Adams, P. J. Edelson, H. S. Koren, Eds. (AcademicPress, New York, 1981), pp 375-395.

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U.S. patent application Ser. Nos. 07/724,509, filed on Jun. 28, 1991,now pending, and 07/331,366, filed on Mar. 31, 1989, now abandoned, areincorporated herein by reference.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above-teachings. It is thereforeto be understood that. within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

We claim:
 1. A kit for treating a disease in a patient in need thereof,comprising a catheter and a physiologically acceptable solution,wherein:(i) said catheter is adapted for insertion into a blood vesseland comprises a main catheter body having a balloon element, adapted tobe inserted in said blood vessel and being expansible against the wallsof said vessel so as to hold said main catheter body in place, and meanscarried by said main catheter body for delivering said solution intosaid blood vessel; (ii) said physiologically acceptable solutioncomprises DNA and at least one member selected from the group consistingof heparin, poly-L-lysine, polybrene, dextransulfate, a polycationicmaterial, and bivalent antibodies.
 2. The kit of claim 1, wherein saidphysiologically acceptable solution further comprises a growth factor.3. A kit for treating a disease in a patient in need thereof,comprising(i) a catheter adapted for insertion into a blood vessel,comprising a main catheter body having a balloon element adapted tobeing inserted into said vessel and expansible against the walls of thesaid vessels so as to hold said main catheter body in place in saidvessel and a means carried by said main catheter body for delivering aphysiologically acceptable solution into said blood vessel; (ii) saidphysiologically acceptable solution which may contain an enzyme, milddetergent or lipid; and (iii) a means for causing a cell attached ontothe walls of a vessel or in an organ or tissue in said patient toexpress an exogenous therapeutic agent protein, comprising a formulationadapted for delivery by said catheter for the transfer and uptake of RNAor DNA into said cell attached onto the walls of a vessel or in an organor tissue in said patient.
 4. The kit according to claim 3, wherein saidDNA is antisense DNA.
 5. The kit of claim 3, wherein said solutioncontains, as said enzyme, at least one member selected from the groupconsisting of dispase, trypsin, collagenase, papain, pepsin,chymotrypsin, and lipases.
 6. The kit of claim 3, wherein said solutioncontains at least one member selected from the group consisting ofNonidet P-40, Triton X100, deoxycholate, and sodium dodecyl sulfate. 7.The kit of claim 3, wherein said main catheter body comprises two spacedballoon elements, adapted to be inserted in a blood vessel and bothbeing expansible against the walls of the blood vessel, for providing achamber in said blood vessel and so as to hold said main catheter bodyin place, and whereas said means for delivering a physiologicallyacceptable solution into said chamber is situated in between saidballoon elements.
 8. The kit of claim 3, wherein said means fordelivering said solution into said blood vessel comprises a plurality ofpore means.
 9. The kit of claim 3, wherein said formulation comprises aretrovirus, a plasmid, a liposomal formulation, or a plasmid complexwith a polycationic substance.
 10. The kit of claim 3, wherein saidformulation is a liposomal formulation.